title: Shiga-like toxin binds with high avidity to multivalent O-linked blood group P1 determinants on mucin-type fusion proteins.
abstract: The binding of Shiga-like toxin 1 (Stx1) and Shiga-like toxin 2 (Stx2) to a mucin-like fusion protein, P-selectin glycoprotein ligand-1/mouse IgG2b (PSGL-1/mIgG2b), carrying multiple copies of the blood group P1 determinant on O-glycans was investigated with western blot and the biosensor Biacore. Chinese hamster ovary K-1 (CHO-K1) cells were stably transfected with linearized plasmids encoding the PSGL-1/mIgG2b fusion protein, the pigeon α1,4-galactosyltransferase (α4Gal-T) and the core 2 β1,6-N-acetylglucosaminyltransferase (C2GnT-I). Western blot analyses of purified PSGL-1/mIgG2b and liquid chromatography-mass spectrometry (LC-MS) of released O-glycans confirmed the presence of the P1 determinant. Western blot analysis indicated strong binding of Stx1, but not Stx2, to PSGL-1/mIgG2b. In a Biacore assay, Stx1 and Stx2 were immobilized on a dextran chip and the binding of purified PSGL-1/mIgG2b and a P(k)-albumin neoglycoprotein was analyzed. Stx1 and Stx2 bound with high avidity to both PSGL-1/mIgG2b and P(k)-albumin, while the Stx1 binding was the strongest. In summary, we have shown that the pigeon α4Gal-T can be aberrantly expressed in CHO cells together with the core 2 enzyme to generate multiple, O-linked P1 determinants on a simultaneously expressed mucin-type fusion protein. P1-decorated PSGL-1/mIgG2b bound with high avidity to both Stx1 and Stx2, and as such constitutes a potential therapeutic inhibitor of these toxins.
authors.affiliation: Department of Clinical Chemistry and Transfusion Medicine, Sahlgrenska Academy, University of Gothenburg, SE-41345 Gothenburg, Sweden.
authors.forename: Reeja
authors.initials: R
authors.lastname: Maria Cherian
authors.forename: Stefan
authors.initials: S
authors.lastname: Gaunitz
authors.forename: Anki
authors.initials: A
authors.lastname: Nilsson
authors.forename: Jining
authors.initials: J
authors.lastname: Liu
authors.forename: Niclas G
authors.initials: NG
authors.lastname: Karlsson
authors.forename: Jan
authors.initials: J
authors.lastname: Holgersson
mesh_terms: D000818:Animals; D016466:CHO Cells; D010856:Columbidae; D006224:Cricetinae; D003412:Cricetulus; D005915:Globosides / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051379:Mice; D017351:N-Acetylglucosaminyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011134:Polysaccharides / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D022622:Shiga Toxin 1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D022641:Shiga Toxin 2 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D054323:Shiga-Toxigenic Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D005915:Globosides; D007074:Immunoglobulin G; D008562:Membrane Glycoproteins; C084882:P-selectin ligand protein; D011134:Polysaccharides; D011993:Recombinant Fusion Proteins; D022622:Shiga Toxin 1; D022641:Shiga Toxin 2; C480041:galactopyranosyl-1-4-paragloboside; D017351:N-Acetylglucosaminyltransferases; C053024:beta-1,3-galactosyl-O-glycosyl-glycoprotein beta-1,6-acetylglucosaminyl transferase
keywords: SPR; Shiga-like toxin; blood group P1; mass spectrometry; mucin